Stressor-associated alterations in porcine plasma prolactin

Experiments were conducted to determine effects of restraint and thermal stressors on plasma prolactin (PRL) in castrated male pigs. A single 20-min restraining period in a restraining cage which prevented both movement and injury increased (P less than 0.05) plasma PRL when applied at either 0800 or 1600 hr. Exposure to 32 degrees C at 0800-1000 hr or at 1600-1800 hr produced more moderate increases (P less than 0.05). A combination of 20 min restraint and 2 hr at 32 degrees C produced a response similar to restraint alone. Twenty minutes after stressor application plasma PRL concentrations in pigs exposed to restraint or restraint +32 degrees C at 1600 h were greater (P less than 0.05) than concentrations measured in all other treatment groups at that time interval. However, there were no statistically significant differences in additional quantitative indices of the plasma PRL responses (maximal level, maximal change, or integrated response above basal levels) among restraint, 32 degrees C, or restraint +32 degrees C, nor between morning and afternoon applications of treatment. Such data do not provide, therefore, any strong evidence for stressor-dependent or circadian differences in plasma PRL response. A second study subjected castrated male pigs to 20 degrees C (controls), 20 +/- 12 degrees C (cyclic temperature, sine wave variation), 5 degrees C constant, and 5 +/- 12 degrees C cyclic for 20 days. After 6 days exposure to 5 degrees C constant or 5 +/- 12 degrees C cyclic there were decreases (P less than 0.05) of 59 and 67% respectively in plasma PRL when compared either with pretreatment levels or with levels in pigs at 20 or 20 +/- 12 degrees C. There were no differences in PRL responses between cyclic vs constant temperatures. These results are the first to indicate that plasma PRL in pigs is affected by acute restraint and thermal stressors.     

Water-soluble prolactin receptors from porcine mammary gland

Two types of prolactin receptors were identified in sow mammary gland. When light membranes were prepared on a discontinuous sucrose gradient (0.3 and 1.7 M) and then diluted and washed with 0.3 M sucrose solution, a large amount (about 50%) of receptors were released from membranes and appeared in the supernatant fraction. These two forms (hydrophobic and water-soluble) of receptors were characterized as having the same binding specificity for lactogenic hormones and a similar affinity constant for ovine prolactin (K alpha approximately 10-12 X 10(9) M-1). Polyclonal antibodies and one monoclonal (mAb M110) antibody, obtained against partially purified prolactin receptors from rabbit mammary gland, cross-reacted effectively with sow mammary receptors. They completely inhibited the specific binding of [125I]oPRL to membrane and water-soluble receptors. The present studies indicate that the two types of sow prolactin receptors could represent the same molecular entity and confirm that prolactin receptors from rabbit and sow mammary gland exhibit numerous antigenic similarities.     

Demonstration of a specific receptor for prolactin in porcine granulosa cells.

Porcine granulosa cells and subcellular fractions from these cells have been shown to have a specific receptor for ovine prolactin (OPRL). Ovine growth hormone demonstrated 7% of the potency of OPRL in displacing 125I-OPRL from its binding site; FSH, TSH, LH, insulin and ACTH showed negligible cross-reactivity. Scatchard analysis of the displacement curves suggested that 125I-OPRL has a high affinity for its receptor with a dissociation constant (Kd) for the granulosa cell-receptor of 7.4-7.7 times 10(-10) M with no change as the follicle enlarges. In contrast, the specific binding of prolactin decreased markedly with maturation of the follicles with an apparent decrease in binding sites/cell from 555 in small follicles to 300 in large (preovulatory) follicles. The demonstrated Kd's were within the range of prolactin concentrations easily attained in vivo and were in good agreement with values obtained in our laboratory and elsewhere for the prolactin receptor from mammary gland and other tissues. Consequently, these studies may provide a basis for a better understanding of the role of prolactin in ovarian function.     

Structure of the carbohydrate chain of glycosylated porcine prolactin

Cyanogen bromide and chymotryptic fragments both containing a carbohydrate chain were obtained from the glycosylated form of porcine prolactin. By means of the glycosidase hydrolysis and the Smith degradation the carbohydrate chain of the glycoprotein was shown to be of the "complex" type of N-bound oligosaccharides and identical to the chain of the luteinizing hormone.     

Isolation of prolactin from lyophilized porcine pituitary glands]

The optimal conditions for lyophilization of porcine pituitary glands and isolation of pure prolactin from lyophilized preparation have been investigated. The isolation method consisted in the extraction of crude pituitary preparation with acidified acetone followed by precipitation of crude prolactin preparation (acid acetone powder) by increasing the concentration of acetone in the extract to 92%. Further purification of prolactin was achieved by fractional precipitation at varying pH values and gel filtration on Sephadex G-75 column in a pH 7.5 phosphate buffer. This final procedure resulted in obtaining the monomeric form of prolactin. The identity of the isolated hormone was confirmed by spectrophotometric and radioimmunological methods as well as by polyacrylamide gel electrophoresis.     

Linkage and physical mapping of prolactin to porcine chromosome 7

Comparative mapping studies between human and pig have shown that there is conserved synteny between human chromosome 6 and pig chromosomes 1 and 7, but some gene locations are not well established. Prolactin ( PRL ), an anterior pituitary hormone, has been mapped to human chromosome 6, and has tentatively mapped to pig chromosome 7 using Southern-RFLP analysis with a limited number of meioses. To confirm the assignment of prolactin to porcine chromosome 7 by physical and linkage analysis, pig cDNA and human genomic DNA sequences were used to design pig-specific PCR primers. The primers amplified a fragment of ≈2·8 kb. Two polymorphic restriction sites were identified within this fragment with the restriction endonuclease Bst UI. Prolactin was significantly linked to six markers on the published PiGMaP map of pig chromosome 7. Prolactin was physically mapped using a pig × rodent somatic cell hybrid panel. An analysis of these data placed PRL on pig 7p1·1–p1·2 with 100% concordance and was in complete agreement with the linkage data. Both mapping techniques placed PRL in a conserved order with the loci in the syntenic region of human chromosome 6.     

A novel polymorphism of the porcine prolactin gene (PRL)

Prolactin is a protein hormone playing a role in the maintenance of pregnancy in the pig by action on corpora lutea cells and possibly initiating production of progesterone. The prolactin gene is 10 kb in size and is composed of 5 exons and 4 introns. The present work is a report of the swine PRL gene--comparative DNA sequence analysis and the SNP revealed in the promoter region. Based on the bovine prolactin gene, three primer pairs were designed using the Primer3 on-line software. The overlapping fragments covered about 400 nucleotides of the promoter and 78 nucleotides of exon 1. The fragments were amplified; two of them were sequenced and deposited in the GenBank database (AY341908 and AY905690). All fragments were analyzed using multitemperature SSCP (MSSCP) technique. Only one fragment appeared to show a different MSSCP pattern. The samples of differing MSSCP conformers were sequenced and the C499T transition was identified in the 5'UTR region of the gene. The HphI restriction enzyme appeared to recognize the novel SNP. The alignment for homology analysis was performed with porcine, bovine (X01452) and human (NM_000948) DNA sequences available in GenBank database, using BLAST software. The comparative homology analysis results varied in dependence on the species and functional region of the gene.     

Steroid hormones modulate prolactin binding by cultured porcine granulosa cells.

We have studied the effects of the gonadal steroids — testosterone, 17β-estradiol, progesterone, and 5α-dihydrotestosterone on the prolactin-binding activity of porcine granulosa cells maintained in monolayer culture. Testosterone, estradiol, and progesterone all significantly enhanced prolactin binding (55%, 107%, and 112% above control, respectively). In contrast, the non-aromatizable androgen, 5α-dihydrotestosterone, caused an insignificant suppression of prolactin binding. The anti-androgen, cyproterone acetate, did not influence prolactin binding when used alone, and did not inhibit the effects of testosterone. These data suggest that the stimulatory effects of testosterone may require aromatization to estradiol.     

Ultrastructural immunocytochemical localization of prolactin and growth hormone in the porcine pituitary

Summary The immunocytochemical peroxidase-antiperoxidase technique was used to identify prolactin- and growth hormone-producing cells in the porcine pituitary at the ultrastructural level. The growth hormone-producing cells contain round secretory granules (300 nm to 500 nm in diameter). The prolactin-producing cells can be identified by their distinct round and ovoid secretory granules which vary in size. Most of these cells contain large granules (450 nm to 750 nm in diameter), but some prolactin-producing cells display smaller secretory granules (250 nm to 500 nm). The two hormones were localized exclusively in the secretory granules. Staining for prolactin was observed in round and ovoid granules, as well as in small and polymorphic granules within the Golgi complex. This study confirmed (i) that the two hormones are located in different cells, and (ii) that under normal physiological conditions no one cell can synthesize and store both hormones simultaneously.     

Anti-relaxin and relaxin on prolactin and gonadotropin secretion in vitro from porcine pituitary cells

This study examines the possibility of a feedback interaction between gonadal relaxin and the pituitary by investigating the impact of exogenous relaxin and ablation of endogenous with relaxin anti-relaxin serum on pituitary hormone secretion in vitro. Three wells were assigned to treatments: 0, 100 and 1000 ng ml⁻¹ of relaxin, 1:100, 1:1000 and 1:10000 titer of anti-relaxin. Relaxin significantly enhanced prolactin (PRL) secretion (P < 0.05) in long-term culture but had no effect on luteinizing hormone and follicle stimulating hormone secretion. Relaxin anti-serum stimulated a dose dependent increase (P < 0.05) in gonadotropin secretion at 48, 72 and 96 h. Luteinizing hormone and follicle stimulating hormone increased two-fold in 48 h cultures in response to 1:100 anti-relaxin serum in comparison with untreated controls. Anti-relaxin serum at 1:100 completely suppressed PRL secretion after either 48, 72, and 96 h of culture. At 48 h all levels of anti-relaxin serum completely suppressed PRL secretion. These results indicate that endogenous relaxin may be involved at the adenohypophysial level in modulating gonadotropin and PRL release in the pig.     

Receptor binding and Nb2 cell mitogenic activities of glycosylated vs. unglycosylated porcine prolactin

Purified fractions of glycosylated (pGPrl) and unglycosylated (pUGPrl) porcine prolactin were prepared by affinity chromatography on Concanavalin A-Sepharose. The relative binding activities of these two forms of prolactin for receptors from porcine mammary, adrenal cortex and rabbit mammary, as well as their Nb2 cell mitogenic activity were determined. In both the porcine mammary and adrenal cortex receptor binding assays pGPrl had a 2-3 fold lower activity than pUGPrl. In the rabbit mammary binding assay pGPrl had a about a 5 fold lower activity than pUGPrl. Similarly, pGPrl had only about 20% of the activity of pUGPrl in the Nb2 cell proliferation assay.     

The influence of prolactin (PRL) on progesterone secretion by porcine luteal cells in vitro

Porcine luteal cells were obtained from corpora lutea on the 5th, 13th and 17th days of the estrous cycle. The cells were suspended at a concentration of 5 × 10⁴ cells/ml in Eagle's medium with 2% human serum albumin. These cells were incubated with or without 0.01, 0.1, 1 or 10 μg/ml porcine prolactin. The amount of progesterone in cultures was estimated by a radio-immunological method after 30 min, 3 h and 6 h of culturing.Luteal cells obtained on the 5th day of the estrous cycle and incubated without prolactin secreted 71.24 ± 21.91 ng progesterone/ml of medium, whereas under the influence of prolactin at 0.01, 0.1, 1 and 10 μg/ml, 39.06 ± 13.33, 44.31 ± 12.69, 44.88 ± 16.85 and 51.62 ± 15.01 ng progesterone/ml (P<0.01) were secreted. Luteal cells from the 13th day of the estrous cycle incubated without prolactin secreted on average 70.72 ± 9.21 ng progesterone/ml of medium, whereas under the influence of different prolactin doses 50.75 ± 8.52, 46.54 ± 7.13, 43.30 ± 6.78 and 41.68 ± 7.21 ng progesterone/ml (P<0.01) were secreted.Prolactin did not change progesterone secretion by luteal cells obtained on the 17th day of the estrous cycle. An influence of the incubation time on progesterone secretion by these cells was observed: after 30 min of incubation the cells secreted 8.83 ± 2.95 ng/ml, after 3 h 8.12 ± 2.57 ng/ml and after 6 h 6.86 ± 1.91 ng/ml, irrespective of the amount of PRL added.The results suggest that prolactin plays a role in the luteolysis of the corpus luteum.     

Autocrine prolactin inhibits human uterine decidualization: a novel role for prolactin

Human prolactin (PRL) and its receptor (PRLR) are markedly induced during human uterine decidualization, and large amounts of PRL are released by decidual cells as differentiation progresses. However, the role of PRL in decidualization is unknown. In order to determine whether PRL plays an autocrine role in decidualization, human uterine fibroblast cells that were decidualized in vitro with medroxyprogestrerone acetate (1 microM), estradiol (10 nM), and prostaglandin E(2) (1 microM) were exposed to exogenous PRL and/or the pure PRLR antagonist delta1-9-G129R-PRL. As measured by quantitative PCR, cells that were decidualized in the presence of exogenous PRL (0.25-2 microg/ml) expressed significantly lower levels of mRNA for the genes that encode insulin-like growth factor binding protein 1 (IGFBP1), left-right determination factor 2 (LEFTY2), PRL, decorin (DCN), and laminin alpha 1 (LAMA1), all of which are known to be induced during decidualization. These effects were blocked when the cells were exposed simultaneously to PRL and the PRLR antagonist, which confirms the specific inhibitory action of PRL on the expression of decidualization markers. In addition, cells exposed to the PRLR antagonist alone expressed higher levels of the marker gene mRNAs than cells that were decidualized in control media. Taken together, these results strongly suggest that PRL acts via an autocrine mechanism to regulate negatively the extent of differentiation (decidualization) of human uterine cells.     

Demonstration of mRNAs for oxytocin and prolactin in porcine granulosa and luteal cells. Effects of these hormones on progesterone secretion in vitro

The relative levels of mRNAs for relaxin, prolactin, inhibin and oxytocin have been measured in porcine granulosa as well as luteal cells by hybridisation to single-stranded synthetic DNA. The likelihood of a paracrine function of oxytocin and prolactin in the porcine ovary was inferred from the in vitro effects of both hormones on progesterone secretion of ovarian cells. Both hormones were found to inhibit progesterone secretion of luteal cells. In contrast, only prolactin but not oxytocin stimulated progesterone secretion in granulosa cells.     

Homologous radioimmunoassay for secreted mouse prolactin

A highly specific, homologous radioimmunoassay has been developed for the secreted form of mouse prolactin using hormone isolated and purified from the media from long-term pituitary cultures. Mouse pituitary homogenates and mouse serum give parallel dilution response curves in the assay, and no cross-reaction is seen with either mouse growth hormone or mouse placental lactogen. The assay is sensitive to 40 pg per tube, and the reproducibility and precision of the assay are within acceptable limits. Antiserum generated to secreted mouse prolactin cross-reacts with both the secreted and stored forms of the hormone; however, the secreted form shows greater immunopotency. Secreted prolactin also shows greater immunopotency when compared with stored prolactin using an antiserum generated to the stored form of the hormone.